Alanine-Scanning Mutagenesis of α-Conotoxin GI Reveals the Residues Crucial for Activity at the Muscle Acetylcholine Receptor

Recently, the muscle-type nicotinic acetylcholine receptors (nAChRs) have been pursued as a potential target of several diseases, including myogenic disorders, muscle dystrophies and myasthenia gravis, etc. α-conotoxin GI isolated from Conus geographus selectively and potently inhibited the muscle-type nAChRs which can be developed as a tool to study them. Herein, alanine scanning mutagenesis was used to reveal the structure–activity relationship (SAR) between GI and mouse α1β1δε nAChRs. The Pro(5), Gly(8), Arg(9), and Tyr(11) were proved to be the critical residues for receptor inhibiting as the alanine (Ala) replacement led to a significant potency loss on mouse α1β1δε nAChR. On the contrary, substituting Asn(4), His(10) and Ser(12) with Ala respectively did not affect its activity. Interestingly, the [E1A] GI analogue exhibited a three-fold potency for mouse α1β1δε nAChR, whereas it obviously decreased potency at rat α9α10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with α1β1δε nAChR, and Tyr(11) of GI is a critical residue binding with three hydrophobic amino acids of the δ subunit, including Leu(93), Tyr(95) and Leu(103). Our research elucidates the interaction of GI and mouse α1β1δε nAChR in detail that will help to develop the novel analogues of GI.